The present invention relates to methods and systems, as well as assay kits, for the determination or screening of analytes in liquid samples, based on label-free determination of binding events at a solid phase surface.
A wide variety of techniques for determining the concentration of a compound, usually referred to as analyte, in a liquid sample, are based on the binding of the analyte to a ligand immobilised on a solid support, below referred to as solid phase assays. Usually, the assays are of either sandwich or competitive type.
In a competitive type assay, analyte or an analyte analogue is allowed to compete with the analyte for the binding to the immobilised ligand, whereas in a sandwich type assay, a specific reagent is bound to the analyte, either before or after the analyte is bound to the immobilised ligand. While labelling with a marker is usually used to permit detection of the binding events, so-called label-free methods are also known, for example such based on surface plasmon resonance (SPR).
It has been desired in solid phase assays to provide a regeneratable surface of general character, i.e. a surface that easily permits the immobilized ligand to be removed and be replaced by binding a different ligand to the surface.
One type of such a surface is disclosed in U.S. Pat. No. 4,271,140, where a complex of the formula ABL(BL)nA1 is adsorbed onto the surface. In the complex, BL is a binding ligand, n is at least 1, ABL is a receptor specific for BL, A1 is a receptor for the analyte being assayed, BL is covalently bonded to A1, and ABL is reversibly bonded to BL. Thus, a single surface coated with ligand binding receptor ABL may be used for different assays by reversibly binding and releasing different conjugates (BL)nA1 to the surface. This is a particular advantage in automated flow assay equipment. In a competitive specific binding assay, for example, based on this concept, the analyte and a labelled analyte analogue will compete for the binding to the immobilised conjugate (BL)nA1. A disadvantage of the above approach is, however, that in the conjugate (BL)nA1, the covalent binding of receptor A1 to the binding ligand BL may have a negative influence on the receptor function.
The use of a universal support in a solid phase assay is also described in WO 95/24649. In this assay, one or more binding agents are provided with tail groups which can bind specifically to one or more capture agents immobilised on the support. The binding agent is usually an antibody, and the tail groups and the respective capture agents are complementary oligonucleotide sequences which can hybridise to each other. The assay is performed by sequentially or simultaneously contacting the sample with the binding agent(s) and the immobilised capture agent(s), and determining the fraction of binding sites of a binding agent occupied by the analyte to determine the concentration of the analyte in the sample.
A similar approach is described in WO 96/06948, where double stranded nucleic acid duplexes serve as universal cleavable link systems in different types of immunoassays. In an embodiment of competitive assay, one oligonucleotide is bound to the analyte or to an analogue thereof which is allowed to compete with the target analyte for a labelled immunoreagent. The second oligonucleotide is bound to a solid support. The analyte competitor will bind competitively with free analyte to the immunoreagent. When the reaction solution is contacted with the solid support under hybridising conditions, the oligonucleotide-coupled analyte or analyte analogue will bind to the support via an oligonucleotide duplex. By determining the concentration of label in the duplexes, the concentration of analyte may be determined.
A disadvantage common to all the above described methods is that they require some type of labelling.
Recently, label-free surface sensitive measuring techniques have been developed for measuring and quantifying biomolecular interactions. In these techniques, a receptor capable of binding to an analyte of interest is immobilised to a sensor surface, and binding of the analyte to the receptor is detected as a resulting change of a property of the sensor surface, such as a change in surface mass or surface refractive index. One type of such mass-sensing apparatus uses the phenomenon of surface plasmon resonance (SPR) to study the binding of analytes to receptors immobilized on the sensor surface. The apparatus and theoretical background are described in the literature (see e.g. Jxc3x6nsson, U., et al., BioTechniques 11:620-627 (1991)).
However, for detecting a binding event at a mass detecting sensor surface, the species binding to the surface must be rather large, i.e have a relatively high molecular weight, as the binding of a small molecule to the surface will usually not give a sufficient mass change to be detectable. Prior art assay formats based on the use of a label are therefore in general not readily transformable to a label-free assay format based on mass detection at a sensor surface.
The object of the present invention is therefore to provide novel assay methods and systems which use a regeneratable, or rechargeable, solid support surface of universal character, and which are based on surface mass-sensing and therefore label-free.
Accordingly, in a first aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) providing a conjugate comprising (i) the analyte, or an analyte competitor, and (ii) the other member of said specific binding pair,
c) simultaneously or sequentially contacting a liquid sample containing or suspected of containing the analyte (with a specific binding partner to the analyte) and with said conjugate, said binding partner having a molecular weight of at least five times that of said conjugate,
d) contacting the reacted liquid sample with the solid support to bind reacted and unreacted conjugate to the solid support,
e) determining by mass-sensing at the solid support the amount of analyte binding partner that has bound in step d) to the solid support via said conjugate to determine therefrom analyte in the sample, and
f) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a second aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) providing a conjugate between (i) the analyte, or an analyte competitor, and (ii) the other member of said specific binding pair,
c) contacting said conjugate with said solid support to bind conjugate thereto,
d) contacting a liquid sample containing or suspected of containing the analyte with a specific binding partner to the analyte, said binding partner having a molecular weight of at least five times that of said conjugate,
e) contacting the reacted liquid sample with the solid support having said conjugate immobilised thereto to bind unreacted analyte binding partner thereto,
f) determining by mass-sensing at the solid support the amount of analyte binding partner that has bound in step e) to the solid support via said conjugate to determine therefrom analyte in the sample, and
g) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a third aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) providing a conjugate between (i) the analyte, or an analyte competitor, and (ii) the other member of said specific binding pair,
c) simultaneously or sequentially contacting a liquid sample containing or suspected of containing the analyte with a specific binding partner to the analyte and with the conjugate,
d) contacting the reacted liquid sample with the solid support to bind reacted and unreacted conjugate to the solid support,
e) contacting the solid support with a reagent which specifically binds to said binding partner, said reagent having a molecular weight of at least five times that of a complex between binding partner and conjugate,
f) determining by mass-sensing at the solid support the amount of said reagent that has bound in step d) to analyte binding partner bound to the solid support via said conjugate to determine therefrom analyte in the sample, and
g) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a fourth aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobolized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) providing a conjugate between (i) the analyte, or an analyte competitor, and (ii) the other member of said specific binding pair,
c) contacting said conjugate, with said solid support to bind conjugate thereto,
d) contacting a liquid sample containing or suspected of containing the analyte with a specific binding partner to the analyte,
e) contacting the reacted liquid sample with the solid support to bind unreacted binding partner to conjugate immobilised on the solid support,
f) contacting the solid support with a reagent which specifically binds to said binding partner, said reagent having a molecular weight of at least five times that of a complex of binding partner and conjugate,
g) determining by mass-sensing at the solid support the amount of said reagent that has bound in step f) to analyte binding partner bound to the solid support via said conjugate to determine therefrom analyte in the sample, and
h) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a fifth aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobilized thereto one member of a first specific binding pair capable of reversibly binding to the other member of said first specific binding pair,
b) providing a conjugate comprising (i) the analyte, or an analyte competitor, (ii) the other member of said first specific binding pair, and (iii) one member of a second specific binding pair,
c) simultaneously or sequentially contacting a liquid sample containing or suspected of containing the analyte with a specific binding partner to the analyte and with said conjugate,
d) contacting the reacted liquid sample with the solid support to bind unreacted conjugate to the solid support, complex formation between conjugate and specific binding partner preventing conjugate from binding to the support,
e) contacting the solid support with the other member of said second specific binding pair to permit said other member to bind to the conjugate, said other member having a molecular weight of at least five times that of said conjugate,
f) determining by mass-sensing at the solid support the amount of said other member that has bound in step e) to said conjugate bound to the solid support to determine therefrom analyte in the sample, and
g) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said first specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a sixth aspect, the present invention provides a method of determining an analyte in a liquid sample, comprising the steps of:
a) providing a solid support having immobilized thereto one member of a first specific binding pair capable of reversibly binding to the other member of said first specific binding pair,
b) providing a conjugate comprising (i) the analyte, or an analyte competitor, (ii) the other member of said first specific binding pair, and (iii) a carrier having a molecular weight of at least 5,000 daltons,
c) simultaneously or sequentially contacting a liquid sample containing or suspected of containing the analyte with a specific binding partner to the analyte and with said conjugate,
d) contacting the reacted liquid sample with the solid support to bind unreacted conjugate to the solid support, complex formation between conjugate and specific binding partner preventing conjugate from binding to the support,
e) determining by mass-sensing at the solid support the amount of said conjugate that has bound in step d) to the solid support to determine therefrom analyte in the sample, and
f) regenerating the solid support by subjecting the surface thereof to conditions which disrupt the binding between the members of said first specific binding pair to release the conjugate from the solid support and prepare the surface for a subsequent determination.
In a seventh aspect, the present invention provides an assay kit for an analyte, comprising:
a) a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) a specific binding partner to the analyte, which binding partner is unlabelled,
c) a conjugate between (i) the analyte, or an analyte analogue, and (ii) the other member of said specific binding pair, which conjugate binds competitively with free analyte to said binding partner,
said specific binding partner in b) having a molecular weight of at least five times that of the conjugate.
In an eighth aspect, the present invention provides an assay kit for an analyte, comprising:
a) a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of the specific binding pair,
b) a specific binding partner to the analyte, which binding partner is unlabelled,
c) a conjugate comprising (i) the analyte, or an analyte analogue, and (ii) the other member of said specific binding pair, which conjugate binds competitively with free analyte to said binding partner, and
d) a reagent which binds specifically to said binding partner, which reagent is unlabelled,
said reagent in d) having a molecular weight of at least five times that of a complex between conjugate and binding partner.
In a ninth aspect, the present invention provides an assay kit for an analyte, comprising:
a) a solid support having immobilized thereto one member of a first specific binding pair capable of reversibly binding to the other member of said first specific binding pair,
b) optionally, a specific binding partner to the analyte, which binding partner is unlabelled,
c) a conjugate comprising (i) the analyte, or an analyte analogue, (ii) the other member of said first specific binding pair, and (iii) one member of a second specific binding pair, which conjugate binds competitively with unconjugated analyte to said binding partner, and
d) the other member of said second specific binding pair, which member is unlabelled,
said other member in d) having a molecular weight of at least five times that of the conjugate.
In a tenth aspect, the present invention provides an assay kit for an analyte, comprising:
a) a solid support having immobilized thereto one member of a specific binding pair capable of reversibly binding to the other member of said specific binding pair,
b) optionally, a specific binding partner to the analyte, which binding partner is unlabelled, and
c) a conjugate comprising (i) the analyte, or an analyte analogue, (ii) the other member of said specific binding pair, and (iii) a carrier molecule having a molecular weight of at least 5,000 daltons, which conjugate binds competitively with unconjugated analyte to said binding partner.
In an eleventh aspect, the present invention provides an assay kit for an analyte, corresponding to any one of the assay kits defined above but wherein the conjugate is replaced by a pre-conjugate lacking the analyte or analyte analogue part of the conjugate.
In a twelfth aspect, the present invention provides an assay system for an analyte, which comprises, in addition to any of the assay kits described above, an apparatus for detection by mass-sensing of binding events at the solid support.
In a thirteenth aspect, the present invention provides the use of any one of the method and system aspects described above for concentration analysis, preferably of small molecules, and for drug screening.